IMPROVED CHOLECYSTOKININ-2 RECEPTOR (CCK2R) TARGETING FOR DIAGNOSIS AND THERAPY
20240091390 ยท 2024-03-21
Inventors
Cpc classification
C07K7/02
CHEMISTRY; METALLURGY
A61K51/088
HUMAN NECESSITIES
International classification
A61K51/08
HUMAN NECESSITIES
C07K7/02
CHEMISTRY; METALLURGY
Abstract
The present invention provides valuable peptidomimetics for therapeutic and diagnostic purposes as well as compositions, methods, uses and kits based on these peptidomimetics. In particular, the peptidomimetics of the present invention are incorporated by CCK2R expressing cells, for instance, cancer cells. This allows, for instance, to selectively destroy cancer cells or to selectively image cancer cells that express CCK2R.
Claims
1. A peptidomimetic with the following structure:
X-Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal, wherein X is a chelator comprising a radionuclide or a prosthetic group comprising a radionuclide.
2. The peptidomimetic of claim 1, wherein the chelator or the prosthetic group is bound to a radionuclide selected from the group consisting of: .sup.225Ac, .sup.212Bi, .sup.213Bi, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.69Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.111In, .sup.113 min, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.43Sc, .sup.44Sc, .sup.47Sc, .sup.155Tb, .sup.161Tb, .sup.99mTc, .sup.86Y, .sup.90Y, .sup.169Yb, .sup.175Yb, .sup.52Fe, .sup.169Er, .sup.72As, .sup.97Ru, .sup.203Pb, .sup.212Pb, .sup.51Cr, .sup.52mMn, .sup.89Zr, .sup.105Rh, .sup.166Dy, .sup.166Ho, .sup.153Sm, .sup.149Pm, .sup.151Pm, .sup.172Tm, .sup.121Sn, .sup.117mSn, .sup.142Pr, .sup.143Pr, .sup.198Au, .sup.199Au, .sup.123I, .sup.124I, .sup.125I, Al.sup.18F and .sup.18F.
3. The peptidomimetic of claim 1, wherein the chelator is ##STR00012## wherein the asterisk indicates the position where the chelator is directly bound to the Linker.
4. The peptidomimetic of claim 1, wherein the radionuclide is chelated by the chelator.
5. The peptidomimetic of claim 1, wherein the radionuclide is bound to the prosthetic group by a covalent bond.
6. The peptidomimetic of claim 5, wherein the radionuclide is a radionuclide of a halogen.
7. The peptidomimetic of claim 1, wherein the peptidomimetic is DOTA-Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal.
8. The peptidomimetic of any one of claims 1 to 7, wherein the Linker is selected from the group consisting of GABA-GABA, GABOB-GABOB or ?DGlu-?DGlu.
9. The peptidomimetic of claim 8, wherein the peptidomimetic is DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal.
10. The peptidomimetic of claim 9, wherein the peptidomimetic comprises a radionuclide that is chelated by DOTA.
11. The peptidomimetic of claim 10, wherein the radionuclide is selected from the group consisting of .sup.225Ac, .sup.212Bi, .sup.213Bi, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.69Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.111In, .sup.113mIn, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.43Sc, .sup.44Sc, .sup.47Sc, .sup.155Tb, .sup.161Tb, .sup.99mTD, .sup.86Y, .sup.90Y, .sup.169Yb, Al.sup.18F and .sup.175Yb.
12. The peptidomimetic of claim 11, wherein the radionuclide is .sup.90Y, .sup.111In, .sup.68Ga, .sup.225Ac or .sup.177Lu.
13. The peptidomimetic of claim 12, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
14. The peptidomimetic of claim 12, wherein the peptidomimetic is .sup.177Lu-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
15. A method of producing the peptidomimetic of any one of claims 1 to 14, comprising synthesizing the peptidomimetic.
16. A pharmaceutical composition comprising the peptidomimetic of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.
17. Use of the peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16 for imaging a tumor.
18. A method of imaging cells, wherein the method comprises the steps of a) contacting the cells with the peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16, thereby bringing the radionuclide in contact with the cells, and b) visualizing the radionuclide that is in contact with the cells.
19. The method of claim 18, wherein contacting comprises administering the peptidomimetic to a patient.
20. The method of claim 18 or 19, wherein the cells express CCK2R on the surface of the cells.
21. The method of any one of claims 18 to 20, wherein the cells are cancer cells.
22. The method of any one of claims 18 to 21, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
23. The peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16 for use in therapy.
24. The peptidomimetic for use according to claim 23, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 or .sup.177Lu-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
25. The peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16 for use in the treatment of a cancer.
26. The peptidomimetic for use according to claim 25, wherein the cancer expresses CCK2R on the surface of cancer cells.
27. The peptidomimetic for use according to claim 25 or 26, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 or .sup.177Lu-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
28. The peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16 for use in diagnosing a cancer.
29. The peptidomimetic for use according to claim 28, wherein the cancer expresses CCK2R on the surface of cancer cells.
30. The peptidomimetic for use according to claim 28 or 29, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
31. A method of treating a patient suffering from a disease, the method comprising administering to the patient the peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16.
32. The method of claim 31, wherein the disease is a cancer.
33. The method of claim 32, wherein the cancer expresses CCK2R on the surface of cancer cells.
34. The method of any one of claims 31 to 33, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 or .sup.177Lu-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
35. A method of diagnosing cancer in a patient, wherein the method comprises the steps of a) contacting a cancer cell of the patient with the peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16, thereby bringing the radionuclide in contact with the cancer cell, and b) visualizing the radionuclide that is in contact with the cancer cell.
36. The method of claim 35, wherein the cancer expresses CCK2R on the surface of cancer cells.
37. The method of claim 35 or 36, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 or .sup.177Lu-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
38. Use of the peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16 for diagnosing cancer.
39. Use of the peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16 for treating cancer.
40. Use of the peptidomimetic of any one of claims 1 to 14 or the pharmaceutical composition of claim 16 for distinguishing a cancer cell from a healthy cell.
41. The use of any one of claims 38 to 40, wherein the cancer expresses CCK2R on the surface of cancer cells.
42. The use of any one of claims 38 to 41, wherein the peptidomimetic is .sup.68Ga-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 or .sup.177Lu-DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0036] The invention will be described in more detail in the following section and illustrated in accompanying figures showing in:
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DETAILED DESCRIPTION OF THE INVENTION
[0049] All publications, including but not limited to patents, patent applications and scientific publications, cited in this description are herein incorporated by reference for all purposes as if each individual publication were specifically and individually indicated to be incorporated by reference.
[0050] The use of the term comprising as well as other grammatical forms such as comprises and comprised is not limiting. The terms comprising, comprises and comprised should be understood as referring to an open-ended description of an embodiment of the present invention that may, but does not have to, include additional technical features in addition to the explicitly stated technical features. In the same sense, the term involving as well as other respective grammatical forms such as involves and involved is not limiting. The same applies for the term including and other grammatical forms such as includes and included. Section headings throughout the description are for organizational purposes only. In particular, they are not intended as limiting for various embodiments described therein, and it is to be understood that embodiments (and features therein) described under one subheading may be freely combined with embodiments (and features therein) described under another subheading. Further, the terms comprising, involving and including, and any grammatical forms thereof, are not to be interpreted to exclusively refer to embodiments that include additional features to those explicitly recited. These terms equally refer to embodiments that consist of only those features that are explicitly mentioned.
[0051] As used herein, the term peptidomimetic, peptide analogue or peptide derivative or peptide conjugate refers to a compound that comprises a polymer of two or more amino acids that comprises at least one unnatural amino acid, pseudopeptide bond or chemical moiety that is different from an amino acid, such as a reporter group or a cytotoxic group, including a chelator, a prosthetic group, a Linker or a pharmacokinetic modifier. In particular, the peptidomimetics of the invention comprise group X, wherein X is a chelator comprising a radionuclide or a prosthetic group comprising a radionuclide, and a Linker. A peptidomimetic as defined herein generally mimics a biological activity of a natural peptide. In the present case, the peptidomimetic of the present invention mimics the ability, in the sense of having the ability, of natural CCK2R ligands, such as gastrin, to specifically bind to CCK2R.
[0052] As used herein, the term amino acid polymer refers to a polymer of two or more amino acids.
[0053] The term amino acid as used herein refers to a compound that contains in its monomeric state at least an amine (NH.sub.2) and a carboxyl (COOH) functional group. Two amino acids can be covalently bonded to one another by a peptide bond. If an amino acid is conjugated to another amino acid through a pseudopeptide bond, as described below, the amine or carboxyl group may be replaced by other chemical moieties depending on the nature of the pseudopeptide bond. Preferably, the term amino acid as used herein refers to alpha- or beta-amino acids. As used herein the term amino acid includes the proteinogenic amino acids alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); valine (Val) and selenocysteine (Sec). The term amino acid as used herein also includes unnatural amino acids.
[0054] Unnatural amino acids in the sense of the present application are non-proteinogenic amino acids that occur naturally or are chemically synthesized, for example, norleucine (Nle), methoxinine, homopropargylglycin, ornithine, norvaline, homoserine, and other amino acid analogues such as those described in Liu C C, Schultz P G, Annu Rev Biochem 2010, 79: 413-444 and Liu D R, Schultz P G, Proc Natl Acad Sci U S A 1999, 96: 4780-4785. An unnatural amino acid, as used herein, may be, for instance, a proteinogenic amino acid in D-form, for instance, DGIu. Additional contemplated unnatural amino acids are para-, ortho- or meta-substituted phenylalanine, such as para-ethynylphenylalanine, 4-Cl-phenylalanine (Cpa), 4-amino-phenylalanine, and 4-NO.sub.2-phenylalanine, homoprolin, homoalanine, beta-alanine, 1-naphthylalanine (1Nal), 2-naphtylalanine (2Nal), p-benzoyl-phenylalanine (Bpa), biphenylalanine (Bip), homophenylalanine (hPhe), homopropargylglycine (Hpg), azidohomoalanine (Aha), cyclohexylalanine (Cha), aminohexanoic acid (Ahx), 2-aminobutanoic acid (Abu), azidonorleucine (Anl), tert-leucine (Tle), 4-amino-carbamoyl-phenylalanine (Aph(Cbm)), 4-amino-hydroorotyl-phenylalanine (Aph(Hor)), S-Acetamidomethyl-L-cysteine (Cys(Acm)), 3-benzothienylalanine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta). Some of these unnatural amino acids have been explored already for somatostatin and bombesin analogues (Fani M et al., J Nucl Med 2011, 52: 1110-1118; Ginj M et al., Proc Natl Acad Sci U S A 2006, 103: 16436-16441; Ginj M et al., Clin Cancer Res 2005, 11: 1136-1145; Mansi R, J Med Chem 2015, 58: 682-691).
[0055] As used herein, the term hydrophobic amino acid refers to amino acids that have a net zero charge at physiological pH (about pH 7.4). Hydrophobic amino acids can be proteinogenic hydrophobic amino acids or unnatural hydrophobic amino acids. Proteinogenic hydrophobic amino acids are for example serine, threonine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan. Preferred proteinogenic hydrophobic amino acids are proline, isoleucine, leucine, phenylalanine, tyrosine and tryptophan. Unnatural hydrophobic amino acids are, for example, norleucine (Nle), methoxinine, tert-leucine (Tle), 1-naphthylalanine (1Nal), 2-naphtylalanine (2Nal), 3-benzothienylalanine, p-benzoyl-phenylalanine (Bpa), biphenylalanine (Bip), homophenylalanine (hPhe), homopropargylglycine (Hpg), azidohomoalanine (Aha), cyclohexylalanine (Cha), aminohexanoic acid (Ahx), 2-aminobutanoic acid (Abu), azidonorleucine (Anl), 2-aminooctynoic acid (Aoa), norvaline (Nva), para-ethynylphenylalanine, 4-Cl-phenylalanine, homoproline and homoalanine.
[0056] In some embodiments the peptidomimetic of the present invention has a cellular uptake (i.e. binding to the cell membrane and internalization into the cells) to a degree of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the total activity in an assay as described in Example 2.
[0057] In some embodiments, the peptidomimetic of the present invention specifically binds to CCK2R. The binding affinity of the peptidomimetic of the invention can be at least about 2%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of the binding affinity of CCK8, minigastrin or pentagastrin for CCK2R. In some embodiments of the present invention the binding affinity of the peptidomimetic can even be higher than the binding affinity of CCK8, minigastrin or pentagastrin for CCK2R, for example, at least about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 500%, about 1000%, about 1500%, or about 2000% of the binding affinity of CCK8, minigastrin or pentagastrin for CCK2R in an assay as described in Example 4 of WO 2018/224665 A1.
[0058] Specific binding to CCK2R in the context of the present invention means binding of the peptidomimetic of the present invention to CCK2R that can be displaced with a cognate ligand of CCK2R, such as gastrin, or a radiolabelled variant of a cognate ligand, such as [.sup.125I]Tyr.sup.12-labelled gastrin-I. The half-maximal inhibitory concentration (IC50) is a measure of this specific binding as explained above and can be obtained from an assay as described in Example 4 of WO 2018/224665 A1.
[0059] Binding affinity according to the present invention is determined by measuring the half maximal inhibitory concentration (1050), wherein binding affinity and IC50 value have an inverse relationship, meaning that binding affinity increases with decreasing IC50 value and binding affinity decreases with increasing 1050 value. Therefore, a binding affinity of about 50% means that the IC50 value is about twice as high as the IC50 value of CCK8, minigastrin or pentagastrin for CCK2R.
[0060] The term charged amino acid as used herein includes amino acids that have non-zero net charge at a physiological pH of about 7.4 and includes proteinogenic and unnatural amino acids, for instance, the proteinogenic amino acids Arg, Lys, His, Glu, and Asp.
[0061] The structure of the peptidomimetic is indicated in the three-letter amino acid code known to the person skilled in the art, starting with the N-terminus (amino terminus) of the amino acid sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal of the peptidomimetic on the left and ending with the C-terminus of the peptidomimetic on the right. For example, if the Linker in the structure X-Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal, e.g. DOTA-Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal, does not comprise an amino acid, the amino acid ?Ala forms the N-terminus of the peptidomimetic. In the structure X-Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal, 1Nal forms the C-terminus, which may be preferably amidated.
[0062] The chemical bond that connects two amino acids of the peptidomimetic of the present invention, such as of the amino acids in the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal, may be a peptide bond, i.e., an amide bond (CONH). A peptide bond as used herein can be formed between an amino group attached to the alpha carbon of one amino acid and the carboxyl group attached to the alpha carbon of another amino acid. A peptide bond may also form between an amino group and a carboxyl group, one of which is not attached to the alpha carbon of the amino acid, but to the side chain of the amino acid (isopeptide bond), for example the amino group in the side chain of lysine. The chemical bond that connects two amino acids may also be a pseudopeptide bond. In preferred embodiments, the chemical bond that connects two amino acids of the peptidomimetic () is an amide bond, unless otherwise indicated.
[0063] The term pseudopeptide bond as used herein refers to a bond that connects two amino acids and is not an amide bond (CONH). A pseudopeptide bond may also be included in the amidated C-terminus. Any pseudopeptide bond known in the art is contemplated in the context of the present invention, for instance, CH.sub.2NH, CONRCH.sub.2, CONCH.sub.3 (the latter also referred to as N-Me), or CONR, wherein R is alkyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1-methyl-2-methylpropyl, 2,2-dimethylpropyl, or 1-ethylpropyl.
[0064] Herein, the identity of the chemical bond between two amino acids may be indicated in parentheses or square brackets in between the amino acids that are connected through the bond, for instance, Trp-(NMe)Nle. In this case, the two amino acids Trp and Nle are connected by a pseudopeptide bond of the structure CONCH.sub.3, wherein the carboxyl group of Trp reacted with the amino group of Nle in a condensation reaction under release of one molecule water and the resulting amide nitrogen is methylated. The following spellings, exemplarily given for two amino acids Trp and Nle and the pseudopeptide bond N-Me, are interchangeably used herein to indicate the nature of the pseudopeptide bond: Trp-(N-Me)-Nle, Trp-(N-Me)Nle and Trp(N-Me)-Nle. Alkylester, alkylether and urea bonds are also contemplated as pseudopeptide bonds. Other pseudopeptide bonds such as 1,2,3-triazoles (Mascarin A et al., Bioconjug Chem 2015, 26: 2143-2152) or other amide bond bioisosters, which have shown to stabilize peptidomimetics and improve tumour targeting are also contemplated.
[0065] In some embodiments the presence of a pseudopeptide bond is indicated by the term psi, as commonly used in the art. For instance, X.sup.4-psi[CH.sub.2NH]-X.sup.5 indicates that the two amino acids X.sup.4 and X.sup.5 are connected via the pseudopeptide bond CH.sub.2NH. In some embodiments the pseudopeptide bond can be -psi[CH.sub.2NHCONH], -psi[CH.sub.2NH], -psi[CH.sub.2CH.sub.2], -psi[CSNH] or -psi[Tz]-.
[0066] In some embodiments the pseudopeptide bonds are: COO, COS, COCH.sub.2, CSNH, CH.sub.2CH.sub.2, CHCH, CC, NHCO, CH.sub.2S, CH.sub.2NHCONH and CH.sub.2O.
[0067] In the context of the present invention L- and D-amino acids are equally contemplated. Any amino acid of the present invention may be present in L- or D-form unless otherwise stated. The L-from is indicated by reciting a L directly before the name of the amino acid, the D-form is indicated by reciting a D directly before the name of the amino acid. For instance, DGIu refers to the D-form of the amino acid glutamate and LGlu refers to the L-form of the amino acid glutamate. In preferred embodiments, the enantiomeric form of one, more or all of the amino acids of the peptidomimetic is the L-form. For instance, if the nomenclature encompasses both enantiomeric forms, for example, as is the case in Asp, then the preferred enantiomeric form is the L-form (as in LAsp).
[0068] The term chelator is used as in the art and refers to organic compounds that are polydentate ligands that form two or more coordinate bonds with a metal atom.
[0069] In preferred embodiments of the present invention the C-terminus (carboxy terminus) of the peptidomimetic is modified to, for example, reduce proteolytic degradation, increase shelf life, and/or improve cellular uptake. The C-terminus may be for example amidated with an NRR group, wherein R and R are independently hydrogen or a substituted or non-substituted alkyl as defined herein. In some embodiments R and R are independently ethyl, propyl, butyl, pentyl or hexyl. In a preferred embodiment the peptidomimetic of the present invention is amidated at the C-terminus with a NH.sub.2 group. The C-terminus can also be modified with an ester of the type C(O)OR, wherein R can be a substituted or non-substituted alkyl as defined herein, for instance, ethyl, propyl, butyl, pentyl or hexyl.
[0070] The term alkyl as used herein refers to a straight-chain or branched saturated aliphatic hydrocarbon with 1 to 20 (C1-C20), preferably 1 to 15 (C1-C15), more preferably 1 to 10 (C1-C10), and most preferably 1 to 5 (C1-C5) carbon atoms. For example, alkyl may refer to methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1-methyl-2-methylpropyl, 2,2-dimethylpropyl, and 1-ethylpropyl.
[0071] The alkyl group may be substituted with, for example, halogens, such as fluorine, chlorine and bromine, amines, such as a primary amine (NH.sub.2), primary amide, hydroxyl (OH), further oxygen-, sulfur- or nitrogen-containing functional groups, heterocycles, or aryl substituents, such as phenyl and naphthyl.
[0072] The peptidomimetic of the present invention comprises 5 to 10 amino acids. The term comprises 5 to 10 amino acids as used herein means that the peptidomimetic does not have more than 10 amino acids and not less than 5 amino acids.
[0073] Particularly preferred are peptidomimetics that comprise 5, 6, 7, 8, 9 or 10 amino acids. More preferred are peptidomimetics that comprise 7 amino acids.
[0074] In some embodiments of the present invention the peptidomimetic comprises the chelator DOTA that coordinates a radionuclide, for example a metal radionuclide, such as 225 AC, .sup.212Bi, .sup.213Bi, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.69Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.111In, .sup.113mIn, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.43Sc, .sup.44Sc, .sup.47Sc, .sup.155Tb, .sup.161Tb, .sup.99mTc, .sup.86Y, .sup.90Y, .sup.169Yb, or .sup.175Yb.
[0075] In particularly preferred embodiments of the present invention, the DOTA group of the peptidomimetic coordinates the radionuclide .sup.90Y, .sup.111In, .sup.68Ga or .sup.177Lu.
[0076] In some embodiments of the present invention, the radionuclide has no therapeutic effect, such as a cytotoxic effect. In some embodiments, the radionuclide does at least not have a cytotoxic effect to a degree that is therapeutically relevant. The skilled person is able to determine an administered dose/radioactivity dose of the radionuclide that is sufficient for being detected, for instance in a method of imaging, but low enough for not having a therapeutic effect. Consequently, in some embodiments of the present invention, for instance, the method of imaging and any diagnostic uses or methods, a dose of the radionuclide is used that is sufficient for detection, but does not have a therapeutic effect. As used herein, a radionuclide without therapeutic effect is referred to as non-therapeutic radionuclide. Thus, the present invention also relates to non-therapeutic embodiments of the radionuclide. For example, the invention includes non-therapeutic radionuclides.
[0077] Preferred non-therapeutic radionuclides, which can be used for imaging, are .sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.111In, .sup.177Lu, .sup.203Pb, .sup.97Ru, .sup.44Sc, .sup.152Tb, .sup.155Tb, .sup.99mTc, .sup.167Tm, .sup.86Y, and .sup.89Zr.
Cytotoxic group
[0078] In some embodiments, the peptidomimetic comprises a cytotoxic group. The term cytotoxic group as used herein refers to any material or chemical moiety that directly or indirectly causes the death of the cell to which the peptidomimetic that comprises the cytotoxic group is bound or by which it has been internalized.
[0079] The cytotoxic group can be, for example, a chemotherapeutic agent or a radionuclide. If the chemotherapeutic agent or radionuclide that is comprised by the peptidomimetic is internalized by a cell that expresses CCK2R, the CCK2R expressing cell is killed by the chemotherapeutic agent or radionuclide. In some embodiments, the cell to which the peptidomimetic is bound may also be killed without internalizing the peptidomimetic that comprises the cytotoxic group.
[0080] The chemotherapeutic agent can be selected from the group consisting of vinblastine monohydrazide, tubulysin B hydrazide, actinomycin, all-trans retinoic acid, azacitidine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, indotecan, indimitecan, mertansine, emtansine, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, and vinorelbine.
[0081] Radionuclides that can be used include metal and halogen radionuclides. The radionuclide of the present invention can be for example selected from radioisotopes of P, Sc, Cr, Mn, Fe, Co, Cu, Zn, Ga, As, Br, Sr, Y, Tc, Ru, Rh, Pd, Ag, In, Sn, Sb, Te, I, Pr, Pm, Sm, Gd, Tb, Y, Ho, Er, Lu, Ta, W, Re, Os, Ir, Au, Hg, Tl, Pb, Bi, Po, At, Ra, Ac, Th, and Fm. Preferred radionuclides include, but are not limited to, .sup.225Ac, .sup.111Ag, .sup.77As, .sup.211At, .sup.198Au, .sup.199Au, .sup.212Bi, .sup.213Bi, .sup.77Br, .sup.58Co, .sup.51Cr, .sup.67Cu, .sup.152Dy, .sup.159Dy, .sup.165Dy, .sup.169Er, .sup.255Fm, .sup.67Ga, .sup.159Gd, .sup.195Hg, .sup.161Ho, .sup.166Ho, .sup.123I, .sup.125I, .sup.131I, .sup.111In, .sup.192Ir, .sup.194Ir, .sup.196Ir, .sup.177Lu, .sup.189mOs, .sup.32P, .sup.212Pb, .sup.109Pd, .sup.149Pm, .sup.142Pr, .sup.143Pr, .sup.223Ra, .sup.186Re, .sup.188Re, .sup.105Rh, .sup.119Sb, .sup.47Sc, .sup.153Sm, .sup.117mSn, .sup.121Sn, .sup.89Sr, .sup.149Tb, .sup.161Tb, .sup.99mTc, .sup.127Te, .sup.227Th, .sup.201Tl, and .sup.90Y.
Photosensitizer
[0082] In some embodiments of the present invention the peptidomimetic comprises a photosensitizer.
[0083] As used herein the term photosensitizer refers to a material or chemical moiety that becomes toxic or releases toxic substances upon exposure to light such as singlet oxygen or other oxidizing radicals which are damaging to cellular material or biomolecules, including the membranes of cells and cell structures, and such cellular or membrane damage may eventually kill the cells. Photosensitizers as defined herein are known in the art and available to the skilled person. The cytotoxic effects of photosensitizers can be used in the treatment of various abnormalities or disorders, including neoplastic diseases. Such treatment is known as photodynamic therapy (PDT) and involves the administration of a photosensitizer to the affected area of the body, followed by exposure to activating light in order to activate the photosensitizer and convert them into cytotoxic form, whereby the affected cells are killed or their proliferative potential diminished.
[0084] Photosensitizers exert their effects by a variety of mechanisms, directly or indirectly. Thus for example, certain photosensitizers become directly toxic when activated by light, whereas other photosensitizers act to generate toxic species, e.g. oxidizing agents such as singlet oxygen or oxygen-derived free radicals, which are destructive to cellular material and biomolecules, such as lipids, proteins and nucleic acids, and ultimately kill cells.
[0085] In some embodiments the photosensitizer includes, for example, psoralens, porphyrins, chlorins and phthalocyanines. Porphyrin photosensitizers act indirectly by generation of toxic oxygen species and are particularly preferred. Porphyrins are naturally occurring precursors in the synthesis of heme. In particular, heme is produced when iron (Fe.sup.2+) is incorporated in protoporphyrin IX (PpIX) by the action of the enzyme ferrochelatase. PpIX is a highly potent photosensitizer. Further photosensitizer that can be used in the context of the present invention are aminolevulinic acid (ALA), silicon phthalocyanine Pc 4, m-tetrahydroxyphenylchlorin (mTHPC) and mono-L-aspartyl chlorin e6 (NPe6), porfimer sodium, verteporfin, temoporfin, methyl aminolevulinate, hexyl aminolevulinate, laserphyrin-PDT, BF-200 ALA, amphinex and azadipyrromethenes.
Linker
[0086] The peptidomimetic of the present invention comprises an amino acid polymer with the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal. The total number of amino acids comprised by the peptidomimetic is limited as defined herein. In addition to the amino acid polymer ?Ala-Trp-(NMe)Nle-Asp-1Nal, the peptidomimetic of the present invention comprises a Linker and group X as defined herein. X is a chelator comprising a radionuclide or a prosthetic group comprising a radionuclide.
[0087] The Linker connects group X and the amino acid polymer with the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal. Preferably the Linker forms a covalent bond with the sequence 8Ala-Trp-(NMe)Nle-Asp-1Nal as well as with group X. In some embodiments the Linker forms an amid bond with the chelator, the prosthetic group or the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal. In some embodiments the Linker forms an amide bond with the chelator and the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal. In some embodiments the Linker forms an ester bond with group X.
[0088] In some embodiments the Linker forms an amide bond with the amino group of beta-Ala of the amino acid polymer of the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal.
[0089] In some embodiments the Linker is a bifunctional molecule which can form a covalent bond with the amino group of the beta-Ala of the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal on the one side and group X, e.g. DOTA, on the other side under conditions that are amendable to solid phase peptide synthesis.
[0090] In some embodiments the Linker is a small organic molecule with a molecular weight below 1000 Da, below 900 Da, below 800 Da, below 700 Da, below 600 Da, below 500 Da, below 400 Da, below 300 Da, below 200 Da or below 100 Da. In some embodiments, the molecular weight of the Linker is between 50 and 300 Da, between 50 and 400Da, between 50 and 500 Da, between 50 and 600 Da, between 50 and 700 Da, between 50 and 800 Da, between 50 and 900 Da or between 50 and 1000 Da.
[0091] Preferably the Linker does not perturb the specific binding of the amino acid polymer with the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal to CCK2R.
[0092] The Linker can serve as a pharmacokinetic modifier influencing, for example, the hydrophilicity and pharmacokinetics of the peptidomimetic. For instance in some embodiments the Linker may increase the kidney-to-tumour ration of the peptidomimetic.
[0093] The Linker may be a natural or unnatural amino acid, such as Gly, Ala, Gln, Glu, His, all in L- or D-form, or an amino acid polymer consisting of one or more of these amino acids, or any other chemical moiety, such as polyethylene glycol or a carbohydrate, as well as aminoalkanonyl, for instance, aminohexanoyl or aminobenzoyl or piperidine moieties. In some embodiments the Linker can be 6-aminohexanoic acid, 2-aminobutanoic acid, 4-aminobutyric acid, 4-amino-1-carboxymethylpiperidine or urea or another chemical moiety that allows introducing a functional group in the peptidomimetic. Preferred molecules that may be comprised in the Linker are y-amino-butyric acid (GABA), ?-amino-?-hydroxybutyric acid (GABOB) or DGlu.
[0094] In some embodiments the Linker is a combination of the above mentioned Linkers. For instance, the Linker may comprise or consist of two, three, four or five of the above-mentioned molecules, such as amino acids. For instance, the Linker may consist of two molecules 6-aminohexanoic acid, 2-aminobutanoic acid, 4-aminobutyric acid or DGIu. In some embodiments the Linker is further functionalized with hydroxyl-groups or carboxyl-groups to increase the hydrophilicity of the Linker. For instance, the aminoalkanonyl group may be substituted with one or more hydroxyl or carboxyl-groups.
[0095] In some embodiments the Linker is GABOB-GABOB, which refers to two molecules of ?-amino-?-hydroxybutyric acid, which are condensed through an amide bond. In some embodiments the Linker is GABA-GABA, which refers to two molecules of ?-amino-butyric acid, which are condensed through an amide bond. In some embodiments the Linker is gamma-DGlu-gamma-DGlu (?DGlu-?DGlu).
[0096] As used herein, pharmacokinetic modifier means a chemical moiety that influences the pharmacokinetics of the peptidomimetic, such as the hydrophilicity, biodegradation, and clearance. For instance, the pharmacokinetic modifier may increase the half-life of the peptidomimetic in the blood stream.
Chelator and Prosthetic Group
[0097] For the introduction of the radionuclide, the peptidomimetic of the invention comprises a chelator or a prosthetic group. The chelator or prosthetic group is bound to the radionuclide and can be targeted to cells, e.g. cancer cells, such as tumour cells, via the peptidomimetic's affinity for the CCK2R receptor. The chelator or prosthetic group is linked to the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal via the Linker.
[0098] The introduction of the radionuclide into the chelator or prosthetic group can be performed before or after conjugation of the chelator or prosthetic group with the amino acid polymer of the sequence ?Ala-Trp-(NMe)Nle-Asp-1Nal via the Linker.
[0099] In some embodiments the chelator coordinates a metal radionuclide as mentioned herein.
[0100] The chelator may contain different donor groups for metal complexation such as oxygen, nitrogen, sulphur, (carboxyl, phosphonate, hydroxamate, amine, thiol, thiocarboxylate or derivatives thereof) and comprises acyclic and macrocyclic chelators such as polyaminopolycarboxylic ligands.
[0101] In some embodiments the chelator is selected from the group consisting of diethylenetriaminopentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)]phosphinic acid (TRAP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diiacetic acid (NODA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) as well as derivatives thereof such as DOTA or NOTA functionalized with a glutaric acid arm (DOTAGA, NOTAGA). Other chelators are also contemplated in particular chelators for chelating radiometals.
[0102] Further chelators that are contemplated, for example for chelating .sup.99mTc, include, but are not limited to, diamidedithiols (N.sub.2S.sub.2), triamidethiols (N.sub.3S), tetraamines (N.sub.4) and hydrazinonicotinic acid (HYNIC). HYNIC is usually used in combination with co-ligands to complete the coordination sphere of the metal, which include and are not limited to ethylendiamine-N,N-diacetic acid (EDDA) and tricine. In some embodiments, using the organometallic aqua ion .sup.99mTc(CO).sub.3(H.sub.2O).sub.3, tricarbonyl complexes can be generated by exchanging water molecules with mono-, di- and tridentate chelators to form stable complexes including also the click-to-chelate methodology.
[0103] Further chelators, which are for example useful for labelling the peptidomimetics with .sup.68Ga, include, but are not limited to N,N-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N-diacetic acid (HBED-CC), siderophore-based ligands such as desferrioxamine, hydroxypyridinone ligands such as deferiprone and tris(hydroxypyridinone) (THP), and derivatives thereof.
[0104] In some embodiments the chelator is
##STR00002##
wherein the asterisk indicates the position where the chelator is directly bound to the Linker. In some embodiments
##STR00003##
is bound to a nitrogen or oxygen atom of the Linker. For instance, the carbonyl carbon marked with the asterisk may be bound to an amine of the Linker thereby forming an amide bond between the chelator and the Linker. In some embodiments
##STR00004##
is bound to the amine of GABA, GABOB or DGIu of the Linker.
[0105] A preferred chelator is DOTA or derivatives thereof, such as, DO2A, DO3AM.sup.nBu, DO3AM.sup.en, DO3AM.sup.pNO2Bn, Do3AM.sup.C5H12-CO2H, DOTAM, DTMA and DOTA-(gly).sub.4.
[0106] Preferred prosthetic groups of the present invention are labelled with radionuclides of halogens such as iodine, bromine or fluorine, for example those mentioned herein. In some embodiments the prosthetic group will be selected from a group consisting of the Bolton-Hunter reagent, N-succinimidyl-5-(trialkylstannyl)-3-pyridinecarboxylates or N-succinimidyl-4-[.sup.131I]iodobenzoate ([.sup.131I]SIB) for radioiodination. In some embodiments the prosthetic group will be selected from a group comprising but not limited to 4-[.sup.18F]fluorophenacyl bromide, N-succinimidyl-4-[.sup.18F]fluorobenzoate ([.sup.18F]SFB), N-succinimidyl-4-([.sup.18F]fluoromethyl)benzoate, 4-[.sup.18F]fluorobenzaldehyde, 6-[.sup.18F]fluoronicotinic acid tetrafluorophenyl ester ([.sup.18F]F-Py-TFP), silicon-containing building blocks such as N-Succinimidyl 3-(di-tert-butyl[.sup.18F]fluorosilyl)benzoate ([.sup.18F]SiFB), carbohydrate-based prosthetic groups, such as [.sup.18F]fluoro-deoxyglucose, preferably 2-[.sup.18F]fluoro-2-deoxyglucose ([.sup.18F]FDG), and [.sup.18F]fluoro-deoxymannose, preferably [.sup.18F]-fluoro-2-deoxymannose, or derivatives thereof, maleimide-based and heterocyclic methylsulfone-based .sup.18F-synthons, .sup.18F-labelled prosthetic groups such as .sup.18F-azides or .sup.18F-alkynes permitting labelling via click chemistry, .sup.18F-labelled organotrifluoroborates and [.sup.18F]fluoropyridines. In some embodiments a chelator-based labelling approach using aluminum-fluoride (Al.sup.18 F) is applied for radiofluorination.
[0107] In some embodiments, the radiohalide that is bound to the prosthetic group is selected from the group consisting of .sup.123I, .sup.124I, .sup.125I, .sup.131I, and .sup.18F.
Further Aspects and Embodiments of the Invention
[0108] The present invention also relates to a method of producing the present peptidomimetics as described herein. Production of the present peptidomimetics is possible by standard organic chemistry methods and solid phase peptide synthesis methods available to the person skilled in the art. The method at least comprises synthesizing the amino acid polymer of the peptidomimetic (Behrendt R et al., J Pept Sci 2016, 22: 4-27; Jones J, Amino Acid and Peptide Synthesis, Oxford University Press, New York 2002; Goodman M, Toniolo C, Moroder L, Felix A, Houben-Weyl Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics, workbench edition set, Thieme Medical Publishers, 2004).
[0109] The present invention further relates to pharmaceutical compositions, for therapeutic of diagnostic use, comprising the peptidomimetic described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions according to the present invention can be used in the treatment of CCK2R related diseases, such as diseases that are characterized by CCK2R expression or overexpression. In some embodiments the pharmaceutical compositions of the present invention can be used in the treatment of cancer, in particular such cancers that are characterized by expression of CCK2R. In some embodiments of the present invention the pharmaceutical compositions of the present invention can be used to deliver a cytotoxic group, such as a chemotherapeutic agent or a radionuclide to CCK2R expressing tumour cells. Thus, pharmaceutical compositions of the present invention can be used for targeted cancer therapy.
[0110] The present invention also relates to a kit comprising one or more components of the present invention, for instance, the pharmaceutical composition according to the present invention or the peptidomimetic according to the present invention. The kit may further comprise an information leaflet that provides explanations how to prepare or use the peptidomimetic, pharmaceutical composition or diagnostic composition of the present invention. In some embodiments, the kit comprises the pharmaceutical composition of the present invention ready for use. In a further embodiment, the kit comprises two or more compositions that are sufficient to prepare the pharmaceutical composition, ready for use. For instance, in some embodiments, the kit may comprise a first composition comprising a peptidomimetic that comprises a chelator, and a second composition comprising a reporter or cytotoxic group. To prepare the final diagnostic or therapeutic composition, the skilled person would follow the information leaflet provided in the kit and combine the first and second composition to generate a ready to use diagnostic or therapeutic composition.
[0111] The compositions of the present invention can be used for diagnostic purposes. Diagnostic compositions of the present invention can be administered to the patient as part of the diagnostic process, for example, to allow imaging of CCK2R expressing cells or tissues, for example CCK2R expressing tumour cells. The diagnostic composition of the present invention can be used in methods of imaging, such as methods of imaging according to the present invention, for example methods of imaging tumour cells.
[0112] The present invention also relates to a use of the peptidomimetic of the present invention described herein for delivering the radionuclide to cells. Preferably, the radionuclide is delivered to a cell that expresses CCK2R, for example a cancer cell expressing CCK2R. The use of the present peptidomimetic can be in vivo or in vitro. For example, the peptidomimetic of the present invention can be used to deliver a reporter group or a cytotoxic group to a human or animal, for example a mammal, such as mouse, rat, rabbit, hamster or other mammals. In some embodiments of the present invention the peptidomimetic can be used to deliver a reporter group or a cytotoxic group to cells ex vivo, for example, immortalized or primary cell lines that are cultured in cell culture.
[0113] The present invention also relates to a method of imaging cells as described herein. The method of imaging cells described herein makes use of the peptidomimetic described herein. In some embodiments of the present invention, the method of imaging cells makes use of a non-therapeutic peptidomimetic as described herein. The present method of imaging cells may involve or can be based on established methods of imaging, such as computer tomography (CT), magnetic resonance imaging (MRI), scintigraphy, SPECT, PET, or other similar techniques. Based on the individual method of imaging that is used, the skilled person will select the proper radionuclide. The present method of imaging cells can be carried out in vivo or in vitro. In some embodiments of the present invention contacting a cell with the peptidomimetic of the present invention involves administering the peptidomimetic described herein to a patient, for example a patient that suffers from cancer, for example a cancer that involves the expression of CCK2R. In some preferred embodiments the cell is a tumour cell. Thus, in some preferred embodiments a tumour cell is contacted with the peptidomimetic. In some preferred embodiments, the tumour cell expresses CCK2R.
[0114] In some embodiments, the present invention also relates to a method of treating a patient that suffers from a disease that involves the expression of CCK2R, for example a cancer that is characterized by the expression of CCK2R in the tumour cells. Such a method of treating a patient involves the administration of the peptidomimetic of the present invention to the patient.
[0115] In some embodiments, the present invention relates to the peptidomimetic described herein for use in therapy. In preferred embodiments of the present invention the peptidomimetic is for use in the treatment of cancer. Preferably, the cancer is a cancer that expresses CCK2R on the surface of tumour cells.
[0116] The peptidomimetic of the present invention is useful for the diagnostic workup and treatment of various types of cancer, for example: thyroid cancer such as medullary thyroid carcinomas (MTC), lung cancers such as small cell lung cancer (SCLC), gastrointestinal stromal tumours, tumours of the nervous system such as astrocytomas and meningiomas, stromal ovarian cancers, gastrointestinal cancers, neuroendocrine tumours, gastroenteropancreatic tumours, neuroblastomas, tumours of the reproductive system such as breast carcinomas, endometrial carcinomas, ovarian cancers and prostate carcinomas, insulinomas, vipomas, bronchial and ileal carcinoids, leiomyosarcomas, leiomyomas and granulosa cell tumours. In some preferred embodiments the above mentioned types of cancer express CCK2R.
[0117] In some embodiments, the peptidomimetic does not comprise GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2. In some embodiments, the peptidomimetic does not comprise DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2. In some embodiments, the peptidomimetic does not comprise .sup.177Lu-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2. In some embodiments, the peptidomimetic has the structure X-Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal as defined herein, with the proviso that the peptidomimetic does not comprise DOTA-GABOB-GABOB-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2.
[0118] In some embodiments of the invention the peptidomimetic has one of the following structures:
##STR00005## ##STR00006## ##STR00007##
[0119] In some embodiments the peptidomimetic has the structure
##STR00008##
[0120] In some embodiments of the invention the Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal portion of the peptidomimetic is
##STR00009##
that is bound to X via the N-terminal primary amine of the GABOB-GABOB Linker. In a preferred embodiment X is a chelator comprising a radionuclide.
[0121] In some embodiments of the invention the Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal portion of the peptidomimetic is
##STR00010##
that is bound to X via the N-terminal primary amine of the GABA-GABA Linker. In a preferred embodiment X is a chelator comprising a radionuclide.
[0122] In some embodiments of the invention the Linker-?Ala-Trp-(NMe)Nle-Asp-1Nal portion of the peptidomimetic is
##STR00011##
that is bound to X via the N-terminal primary amine of the ?DGIu-?DGIu Linker. In a preferred embodiment X is a chelator comprising a radionuclide.
EXAMPLES
Example 1: Synthesis of Peptidomimetics
[0123] The synthesis of the peptidomimetics of the present invention was performed using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry.
[0124] The peptidomimetics were assembled on a Rink Amide MBHA resin (Novabiochem, Hohenbrunn, Germany) in N,N-Dimethylformamide (DMF) using an excess of Fmoc-protected amino acid, 1-Hydroxy-7-azabenzotriazole (HOAt), and (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) in alkaline medium. The reactive side chains of the amino acids were masked with appropriate protection groups. After assembling the desired amino acid sequence, coupling of DOTA (tris(tBu) ester) was performed followed by cleavage of the peptidomimetic from the resin with concomitant removal of acid-labile protecting groups. After HPLC purification and lyophilization the peptidomimetics were obtained in ?10% yield with a chemical purity ?95% as confirmed by RP-HPLC and MALDI-TOF MS. Radiolabelling of the inventive peptidomimitics with different radiometals was performed using standard radiolabelling protocols by dissolving the peptide in aqueous solution such as 25-50% ethanol or PBS and mixing the solution with an acidic solution such as hydrochloric acid containing the radiometal and a solution such as sodium acetate solution or ascorbic acid solution for pH adjustment and incubating the mixture at high temperature (90-95? C.) for approximately 10-30 min. Radiolabelling with the different radiometals resulted in high labelling yields and radiochemical purity. HPLC analysis of the peptidomimetics and of the radiolabelled derivatives was performed on a Dionex chromatography system consisting of a Dionex UltiMate 3000 Pump (Dionex, Gemering, Deutschland), UV detection at 280 nm (UltiMate 3000 variable UV-detector) and radiodetection (Gabi Star, Raytest, Straubenhardt, Germany) and using a Phenomenex Jupiter 4 ? Proteo 90A 250?4.6 (C12) column and a flow rate of 1 mL/min together with a gradient system of water containing 0.1% TFA (solvent A) and acetonitrile containing 0.1% TFA (solvent B): 0-3 min 10% B, 3-18 min 10-55% B, 18-20 min 80% B, 20-21 min 10% B, 21-25 min 10%.
[0125] The following peptidomimetics (Table 1) were synthesized according to the above method:
TABLE-US-00001 TABLE 1 Name Structure DOTA- DOTA-Gabob-Gabob-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 MGSA DOTA- DOTA-Gaba-Gaba-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 MGSB DOTA- DOTA-?DGlu-?DGlu-?Ala-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 MGSC DOTA- DOTA-DGlu-Ala-Tyr-Gly-Trp-(NMe)Nle-Asp-1Nal-NH.sub.2 MGS5
Example 2: The Peptidomimetics Show High Cellular Internalization
[0126] These studies were carried out according to a previously published protocol (von Guggenberg E et al., Bioconjug Chem 2004, 15: 864-871) using 1.0 million of A431 cells transfected with the plasmid pCR3.1 containing the full coding sequence for the human CCK2R (A431-CCK2R) as well as the same cell line transfected with the empty vector alone (A431-mock) as a control, which were kindly provided by Dr. Luigi Aloj (Aloj L et al., J Nucl Med 2004, 45: 485-494). DMEM supplemented by 1% (v/v) fetal bovine serum was used as internalization medium and the non-specific cell uptake was studied in A431-mock cells instead of performing blocking studies. Additionally, cell internalization assays were performed using 1.5 million of rat pancreatic AR42J cells expressing rat CCK2R. RPMI supplemented by 1% (v/v) fetal bovine serum was used as internalization medium. Blocking experiments were carried out with 1 ?M pentagastrin. The cells were incubated in triplicates with 10,000-500,000 cpm, for example 10,000-60,000 cpm of radiolabelled peptidomimetic (corresponding to a final concentration of 0.4 nM and ?600 fmol of total peptidomimetic in the assay) and incubated at 37? C. for 2-4 h. The internalized fraction in A431-CCK2R and A431-mock cells as well as AR42J cells, was expressed in relation to the total activity added (% of total). For each radiolabelled peptide the mean value of one representative assay performed in triplicates is represented.
[0127] As shown in
Example 3: The Peptidomimetics of the Present Invention have High Affinity to CCK2R
[0128] Affinity for CCK2R was studied in A431-CCK2R cells. The binding affinity was tested in a competition assay against [.sup.125I][3-iodo-Tyr.sup.12,Leu.sup.15]gastrin-I in comparison with pentagastrin (Boc-?-Ala-Trp-Met-Asp-Phe-NH.sub.2) for DOTA-MGSA, DOTA-MGSB and DOTA-MGSC. Radioiodination of gastrin-I was carried out using the chloramine-T method. Non-carrier-added [.sup.125I][3-iodo-Tyr.sup.12,Leu.sup.15]gastrin-I was obtained by HPLC purification and stored in aliquots at ?20? C. Binding assays were carried out in 96-well filter plates (MultiScreen.sub.HTS-FB, Merck Group, Darmstadt, Germany) pretreated with 10 mM TRIS/139 mM NaCl pH 7.4 (2?250 ?L). For the assay a number of 400,000 A431-CCK2R cells per well was prepared in 35 mM HEPES buffer pH 7.4 containing 10 mM MgCl.sub.2, 14 ?M bacitracin and 0.5% BSA (a hypotonic solution disturbing the integrity of the cell membranes). The cells were incubated in triplicates with increasing concentrations of the peptidomimetics (0.0003 to 1,000 nM) and [.sup.125I-Tyr.sup.12]gastrin-I (?25,000 cpm) for 1 h at RT. Incubation was interrupted by filtration of the medium and rapid rinsing with ice-cold 10 mM TRIS/139 mM NaCl pH 7.4 (2?200 ?l) and the filters were counted in the gamma-counter. Half maximal inhibitory concentration (IC.sub.50) values were calculated following nonlinear regression with Origin software (Microcal Origin 6.1, Northampton, MA) and a representative assay was chosen for comparison. As shown in Table 3 a high binding affinity for the CCK2R with IC.sub.50 values in the low nanomolar range could be confirmed for all tested peptidomimetics.
TABLE-US-00002 TABLE 3 Receptor binding of the inventive CCK2R targeting peptidomimetics in comparison with pentagastrin, DOTA-MGSA, DOTA-MGSB and DOTA-MGSC on A431-CCK2R cells as analyzed by displacement with [.sup.125I][3-iodo-Tyr.sup.12, Leu.sup.15]gastrin-I. Tested CCK2R ligand IC.sub.50 [nM] DOTA-MGSA 0.21 ? 0.01 DOTA-MGSB 0.31 ? 0.02 DOTA-MGSC 0.16 ? 0.01 pentagastrin 0.37 ? 0.10
Example 4: Peptidomimetics of the Present Invention Show Improved Biodistribution In Vivo
[0129] Biodistribution studies evaluating the tumour uptake of the radiolabelled inventive CCK2R targeting peptide analogues were performed in xenografted nude mice. All animal experiments were conducted with approval of the national authorities and carried out in compliance with the relevant European, national, and institutional regulations.
[0130] For the induction of tumour xenografts, A431-CCK2R and A431-mock cells were injected subcutaneously in athymic BALB/c nude mice (Charles River or Janvier Labs), respectively (2 million cells in 200 ?L). When tumours had reached a size of approximately 0.2 ml, biodistribution studies were carried out. Groups of 3-5 mice were injected intravenously via a lateral tail vein with the peptidomimetics, labelled with .sup.177Lu (0.5-1 MBq and 0.02-0.03 nmol peptidomimetic) or .sup.68Ga (?0.5 MBq and 0.02-0.03 nmol peptidomimetic). To check for receptor specificity blocking studies using co-injection of a 1000-fold molar excess of the unlabelled peptide (?20 nmol) together with the administration of the .sup.177Lu-labelled peptide were performed (
[0131] In a first study the biodistribution of .sup.177Lu-DOTA-MGSA and .sup.177Lu-DOTA-MGS5 at 4 h p.i. was compared (A431-CCK2R and A431-mock cells inoculated in the right and left flank of 7-week-old female athymic BALB/c nude mice; 5 animals per peptidomimetic). For one mouse in the .sup.177Lu-DOTA-MGSA group the bone sample was too small to measure the radioactivity, and one animal in the .sup.177Lu-DOTA-MGS5 group did not develop the A431-mock xenograft. Results were expressed as percentage of injected activity per gram tissue (% IA/g) and tumour to organ activity ratios were calculated from the activity measured in the dissected tissues (
[0132] In
[0133] As shown in
[0134] The tumour-to-kidney ratio of .sup.177Lu-DOTA-MGSA was increased also when compared to other .sup.177Lu-labelled peptidomimetics (.sup.177Lu-DOTA-MGS8: 9.58?3.84, p<0.02; 177 Lu-DOTA-MGS10: 6.51?2.92, p<0.001; 177 Lu-DOTA-MGS12: 4.52?1.41, p<0.001; 177 Lu-DOTA-MGS11: 7.72?2.26, p<0.002;
[0135] In peptide receptor radionuclide radiotherapy usually multiple therapy cycles are administered to the patient in order to achieve a cumulative absorbed tumour radiation dose of 60 Gy allowing to reach an optimal therapeutic effect. A cumulative dose of <27 Gy for kidneys needs to be met to avoid renal toxicity (Konijnenberg M W et al., EJNMMI Res 201, 4, 47). The radiation dose delivered to the kidneys is a major factor limiting the total amount of radioactivity, which can be administered to patients. Consequently, the kidney uptake of the radiolabelled peptidomimetic influences the cumulative radiation dose, which can be achieved in the tumour lesions. Without being bound by theory, it is believed that the combination of high tumour uptake and improved tumour-to-kidney ratio of the peptidomimetics of the present invention allows administering a higher total amount of radioactivity in targeted radiotherapy reaching a higher absorbed dose in the tumour lesions while limiting the radiation dose delivered to healthy tissue (especially kidneys). No similar report exists on a comparable improvement in tumour-to-kidney ratio of a radiolabelled CCK2R targeting peptide analogue. The peptidomimetics of the present invention therefore display outstanding properties.
Example 5: The Peptidomimetics of the Present Invention have Improved Targeting Properties for Peptide Receptor Radionuclide Therapy
[0136] The biodistribution profile of .sup.177Lu-DOTA-MGSA and .sup.177Lu-DOTA-MGS5 over up to 7 days was studied in female athymic BALB/c nude mice, inoculated with A431-CCK2R cells (left flank) at an age of 6-8 weeks (5 animals per peptidomimetic and for each time point of 1 h, 24 h, 3 d and 7 d). When tumours had reached a size of approximately 0.1-0.2 ml, biodistribution studies were carried out. For both peptidomimetics an overall low non-specific tissue accumulation of radioactivity was observed. In
[0137] When considering the biodistribution over the 7 days, a significant difference was observed in some of the tissues. As shown in
Example 6: The Peptidomimetics of the Present Invention have Improved Targeting Properties for High Sensitivity Imaging
[0138] In further biodistribution studies the tumour uptake and imaging properties of different .sup.68Ga-labelled pepitdiomimetics was evaluated. For this purpose female athymic BALB/c nude mice (Janvier Labs, Le Genest Saint Isle, France) were inoculated with A431-CCK2R cells (left flank) at an age of 8-10 weeks. After a period of approximately 1 week, when tumours had reached a size of 0.1-0.2 ml, the animals were injected intravenously via a lateral tail vein with the peptidomimetics, labelled with .sup.68Ga (?0.5 MBq and 0.02-0.03 nmol peptidomimetic). .sup.68Ga-DOTA-MGSA and .sup.68Ga-DOTA-MGSC both showed a favourable biodistribution profile with fast blood clearance, predominant renal excretion and low unspecific uptake in most tissues (see
[0139] Small animal PET/CT imaging using a dedicated small animal ?PET/CT scanner (Siemens Inveon PET/CT system) at an injected activity of ?5 MBq (corresponding to 0.3 nmol peptide) confirmed the improved targeting properties of .sup.68Ga-DOTA-MGSC over .sup.68Ga-DOTA-MGS5, with best tumour-to-off target ratio allowing for a clear delineation of the tumour xenografts (see
Example 7: The Peptidomimetics of the Present Invention has Increased Stability In Vivo
[0140] To further characterise the stability of the radiolabelled peptidomimetic in vivo, metabolic studies were carried out in 5-6-week-old female BALB/c mice (Charles River, Sulzfeld, Germany) injected intravenously with .sup.177Lu-labelled and .sup.68Ga-labelled peptidomimetics. All animal experiments were conducted in compliance with the Austrian animal protection laws and with the approval of the Austrian Ministry of Science. To allow monitoring of the metabolites by radio-HPLC, mice were injected with a higher amount of radioactivity (20-40 MBq .sup.177Lu, corresponding to ?1 nmol total peptide, or ?10 MBq .sup.68Ga, corresponding to ?1 nmol total peptide) through a lateral tail vein and euthanized by cervical dislocation at selected time points p.i. (10 min for gallium-68 and 30 min for lutetium-177). A sample of blood was collected and the degradation was assessed by radio-HPLC. For this purpose blood samples were precipitated with ACN, centrifuged at 2000 g for 2 min and diluted with water (1:1/v:v) prior to HPLC analysis using a Dionex chromatography system including radiodetection and UV detection equipped with a Phenomenex Jupiter Proteo C12 column (90 ?, 4 ?m, 250?4.6 mm) column using a water/acetonitrile/0.1% TFA gradient system.
[0141] The radiolabelled peptidomimetic of the present invention showed a very high stability against enzymatic degradation in vivo. The percentage of intact .sup.177Lu-DOTA-MGSA present in the blood at 30 min p.i. was 84.4% and compares with other .sup.177Lu-labelled peptidomimetics of the prior art (.sup.177Lu-DOTA-MGS5: 77.0%, .sup.177Lu-DOTA-MGS8: 56.8%, .sup.177Lu-DOTA-MGS10: 86.1%, .sup.177Lu-DOTA-MGS12: 73.3%). At the time point of 10 min p.i. 92.8% of .sup.177Lu-DOTA-MGSA was intact and only 85.9% of .sup.177Lu-DOTA-MGS5. Also for .sup.68Ga-labelled DOTA-MGSA and DOTA-MGSB a high stability in vivo could be observed at 10 min p.i. with 95.1% and 94.7% intact radiopeptide, respectively. For .sup.68Ga-DOTA-MGSC 59.7% intact radiopeptide was observed for the same time point. The stability in vivo resulted to be also considerably increased when compared to .sup.177Lu-labelled PP-F11 and PP-F11 N. [0142] PP-F11: DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH.sub.2 [0143] PP-F11N: DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH.sub.2
[0144] All bonds () in PP-F11 and PP-F11N are amide bonds and all amino acids whose enantiomeric form is not expressly indicated are in the L-form.
[0145] These two peptide derivatives are derived from MG0 by substitution of the penta-Glu sequence with five D-glutamic acid residues and additional substitution of Met with Nle in PP-F11N. The two peptide conjugates were developed with the aim to improve the metabolic stability and pharmacokinetics and were first described in 2012 (Kroselj M et al., Eur J Nucl Med Mol Imaging 2012, 39: S533-S534 and WO 2015/067473 A1). For the same time point at 30 min p.i. .sup.177Lu-PP-F11 and .sup.177Lu-PP-F11N showed values of 5.5 and 12.7% intact radioligand present in the blood, respectively, and resulted to be almost completely degraded. In previous metabolic stability studies investigating the presence of intact radiopeptide in the blood and urine of different .sup.177Lu-labelled MG analogues, including also .sup.177Lu-PP-F-11, 10 min after intravenous injection into BALB/c mice no intact peptide could be found in urine and blood in vivo (Ocak M et al, Eur J Nucl Med Mol Imaging 2011, 38:1426-1435).
[0146] In
[0147] The peptidomimetics of the present invention therefore displays a much higher stability against enzymatic degradation, as compared, for example, with PP-F11 and PP-F11N of the prior art. Surprisingly, the combination of substitutions in different positions, as according to the present invention, allows efficient stabilization of the peptidomimetic.
[0148] Without being bound by any particular theory, it is currently believed that improved in vivo stability may contribute to the improved tumour uptake and retention. The extremely high tumour uptake and tumour retention and most strikingly, the very favourable tumour-to-back-ground activity ratios, especially for kidneys, render the present peptidomimetic particularly useful for diagnostic and therapeutic uses in CCK2R relevant diseases, such as cancer.